Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry
- 3 December 2020
- journal article
- research article
- Published by American Society of Hematology in Blood
- Vol. 136 (23), 2703-2714
- https://doi.org/10.1182/blood.2020005593
Abstract
The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.This publication has 48 references indexed in Scilit:
- Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilisBlood, 2013
- Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIIIOnline Journal of Public Health Informatics, 2012
- C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIIIThe International Journal of Biochemistry & Cell Biology, 2011
- Structural Basis of the Cofactor- and Substrate-Assisted Activation of Human Coagulation Factor IXaStructure, 2009
- The Heparin-Binding Exosite Is Critical to Allosteric Activation of Factor IXa in the Intrinsic Tenase Complex: The Role of Arginine 165 and Factor XBiochemistry, 2007
- The Interface between the EGF2 Domain and the Protease Domain in Blood Coagulation Factor IX Contributes to Factor VIII Binding and Factor X ActivationBiochemistry, 2006
- Structure–Function Relationships in Factor IX and Factor IXaTrends in Cardiovascular Medicine, 2003
- The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 DomainOnline Journal of Public Health Informatics, 2002
- Factor IXa:Factor VIIIa InteractionOnline Journal of Public Health Informatics, 2001
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976